S 593 
1.L5 
Copy 1 



UNIVERSITY OF CALIFORNIA PUBLICATIONS 

IN 

AGRICULTURAL SCIENCES 

Vol. 1, No. 7, pp. 141-172 April 25, 1914 



STUDIES ON AMMONIFICATION IN SOILS 
BY PURE CULTURES 

BY 

C. B. LIPMAN AND P. S. BURGESS 



UNIVERSITY OF CALIFORNIA PRESS 
BERKELEY 



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AGEICULTUBAL SCIENCES. — Charles B. Lipman, Ernest B. Babcock, and John W. 
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VoL 1. 1. The Distribution and Activities of Bacteria in Soils of the Arid 

Eeglon, by Charles B. Lipman. Pp. 1-21. October, 1912 ?0.ao 

2. Studies on the Phenoldisulphonlc Acid Method for Determining Nitrates 

in Soils, by C. B. Lipman and L. T. Sharp. Pp. 23-37. October, 1912. .16 

3. The Effects of Calcium and Magnesium Carbonates on Some Biological 

Transformations of Nitrogen in Soils, by W. P. Kelley. Pp. 39-49. 
December, 1912 — - .10 

4. The Aluminum Seduction Method as Applied to the Determination of 

Nitrates in "Alkali" Soils, by Paul S. Burgess. Pp. 51-62. May, 
1913 ~ - 15 

5. Studies Upon Influences Affecting the Protein Content of Wheat, by G. 

W. Shaw. Pp. 63-126. October, 1913 ...„ - _ _ .75 

6. The Effect of Copper, Zinc, Iron and Lead Salts on Ammonificatlon and 

Nitrification in Soils, by C. B. Lipman and P. S. Burgess. Pp. 127- 

139. March, 1914 15 

7. Studies on Ammonificatlon in Soils by Pure Cultures, by C. B. Lipman 

and P. S. Burgess. Pp. 141-172. April, 1914 25 

Vol. 2. 1. Studies in Juglans I. Study of a New Form of Juglans Calif arnica 
Watson, by Ernest B. Babcock. Pp. 1-46, plates 1-12. December, 
1913 60 

AGBICULTUBE. — The Publications of the Agricultural Experiment Station consist of Bul- 
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360), HI (pp. 400), completed. Volumes IV and V In progress. 
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2. Two new Ascomycetous Fungi Parasitic on Marine Algae, by Minni e 

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thaniel Lyon Gardner. Pp. 165-418; plates 17-27. March, 1903 2.25 

VoL 2. 1. A Eeview of Califomian Polemoniaceae, by Jessie Milliken. Pp. 1- 

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SetcheU. Pp. 115-138; plates 13-14. April, 1905 -. .25 

5. Eegeneration among Kelps, by William Albert Setchell. Pp. 139-168; 

plates 15-17. July, 1905 80 

6. A New Genus of Ascomycetous Fungi, by Nathaniel Lyon Gardner. 

Pp. 169-180; plate 18. July, 1905 „ 15 

7. Teratology in the Flowers of some California Willows, by William 

Warner Mott. Pp. 181-226; plates 16-20. December, 1905 50 

8. 0, 10, 11. (In one cover.) The Resistance of Certain Marine Algae to 

Changes in Osmotic Pressure and Temperature. The R51e of Os- 
motic Pressure in Marine Plants. On the Importance of Physiolog- 
ically Balanced Solutions for Plants. The Antitoxin Action of 
Potassium on Magnesium. By W. J. V. Osterhout. Pp. 227-236. 

March, 1906 - ^ 

12. Cytological Studies in Cyanophyceae, by Nathaniel Lyon Gardner. 

Pp. 237-296; plates 21-26. November, 1906 _ „ 1.00 



V 



UNIVERSITY OF CALIFORNIA PUBLICATIONS 

IN 

AGRICULTURAL SCIENCES 

Vol. 1, No, 7, pp. 141-172 April 25, 1914 



STUDIES ON AMMONIFICATION IN SOILS 
BY PURE CULTURES 

BY 

C. B. LIPMAN AND P. S. BURGESS 



The study of the physiological efficiency of soil bacteria 
rather than their number is admittedly the dominant method in 
soil bacteriological investigations. In view of this fact it is 
singularly striking to note how little work has been accomplished 
in the study of some phases of the physiological efficiency of pure 
cultures of certain groups of soil bacteria. This is especially so, 
since the introduction of radical changes in our soil bacterio- 
logical methods have made it necessary to repeat some, if not all, 
of the work which had been carried out by the old methods. 
However this may be, it remains a fact that, since the publication 
of Marehal's^ splendid work on ammonification in solution 
cultures, but scant information has been adduced from studies 
of soil bacteria which relate to the physiology as well as the 
physiological efficiency of even the more common ammonifying 
bacteria. Certainly, the work carried out along this line in 
direct soil cultures, which recent work has shown to be so far 
superior to solution cultures, has been very meager indeed. 

In order, therefore, to glean some useful information relative 
to the physiological efficiency of pure cultures of a number of 
ammonia-producing bacteria, the writers deemed it wise to select 
a number of organisms and to compare their power to produce 
ammonia not only from one form of organic matter, but from 
several forms, most of which have foiind use in farm prac- 
tice as fertilizers. Accordingly, the following organisms in pure 



142 University of California Publications in Agricultural Sciences [Vol. 1 

culture were selected for the experiments: B. mesentericus vul- 
gatus, Ps. putida, B. vulgatus, B. megatherium, B. mycoides, B., 
suhtilis, B. tumescens, Sarcina lutea, B. profeus vulgaris, B. 
icteroides, B. ramosus, Streptothrix, sp., Ps. fluorescens, B. vul- 
garis (Novy strain), Mic. tefragenus. The organic materials 
experimented with were dried blood (13.16% N), tankage 
(9.62% N), cotton.seed meal (5.5% N), sheep and goat manure 
(2.137o N), peptone (14.14% N), fish guano (8.63% N), and bat 
guano (3.96% N). 

ilETHOD OP Experiments 

Fifty-gram portions of soil were placed in tumblers and 
thoroughly mixed with the organic material to be tested. The 
tumblers were covered with Petri dish covers and sterilized in 
the autoclave at a pressure of thirty pounds for three hours. 
After cooling, the soils were each inoculated with a 1 ec. suspen- 
sion of the organisms to be tested, made up by shaking with some 
sterile water a young slope culture grown on bouillon agar. 
The soil was then stirred with a .sterile spatula after enough 
sterile water had been added to make a moistui-e content in the 
soil about equal to the optimum. The soil cultures thus pre- 
pared were incubated at 28° to 30° C for twelve days. After 
the incubation period the soils were transferred to copper dis- 
tilling flasks, 400 cc. of distilled water and an excess of Mg 
added, and distilled into standard H/10 HCl. The ammonia 
was then determined in the usual way. 

No attempt was made to run all the series with tlie different 
forms or organic matter at the same time, because only the 
relative powers of the different organisms to produce ammonia 
were sought. For the same reason amounts of organic matter 
were chosen in the different series which would least affect the 
physical conditions obtaining in the cultures rather than amounts 
employed which would make the total amount of nitrogen added 
the same in all series. 

For the reasons above given, therefore, the effects of the 
various organisms on any given form of organic material will 
be treated below as a separate series in the ease of each soil and 



1914] Lipman-Burgess : Ammonification in Soils hy Pure Cultures 143 

comparisons between the different series made only where per- 
missible. The data, moreover, are presented so that the dupli- 
cate determinations which were carried out in all cases may be 
compared. The averages of duplicate determinations, however, 
are also given as well as the percentages of nitrogen in the 
organic matter which was transformed to ammonia. 

Three different California soils were tested with each one of 
the ammonifiable materials. The soils were a sandy soil from 
Anaheim, a clay loam from Davis, and a black clay-adobe soil 
from Berkeley. The mechanical and chemical analyses of these 
soils are given in Tables I and II which follow. 







TABLE 


I 






Mechanical Analyses of Soils 




Hyd. value 
Clay 


Sandy 
5.78 


Clay-Loam 
19.12 


Clay-Adobe 
31.93 


0.2.5 mm. 


14.59 


40.93 


25.77 


0..50 




1.04 


3.35 


3.40 


1 




2.30 


6.60 


4.77 


2 




5.28 


7.75 


7.49 


4 




9.62 


8.78 


6.20 


8 




11.58 


8.10 


.87 


16 




4.87 


3.30 


2.78 


32 




15.23 


4.15 


7.66 


64 




29.40 
TABLE 


3.07 
II 


5.44 




Chemical Analysis of Soils 




Insoluble matter 
Soluble silica 


Sandy 
73.59 
11.17 


Clay-Loam 
53.55 
19.77 


Clay-Adobe 
[ 77.84 


K,0 




.64 


.75 


.45 


Na,0 




.15 


.11 


.07 


CaO 




1.39 


.82 


1.05 


MgO 




.93 


1.39 


1.21 


Un,0, 




.04 


.04 


.08 


Pe=03 




5.10 


7.56 


4.68 


AUO3 




3.92 


10.04 


7.79 


PA 




.12 


.13 


.23 


SO3 




.02 


.03 


.08 


Water and 
organic matter 


2.88 


5.62 


5.72 



144 University of California Publications in Agricultural Sciences [Vol. 1 

The pure cultures of the organisms employed in these investi- 
gations were obtained by one of us from the bacteriological 
laboratories of the University of Illinois. Fresh strains of 
B. subtilis and B. mycoides were, however, isolated by us from 
California soils in order to check the stock cultures of the same 
organisms. The same relative powers of producing ammonia 
were, however, found to obtain with the fresh as with the old, 
and different strains thus strengthening the validity of the 
results below discussed. 

Series I. Experiments with Dried Blood 
Sandy Soil 

Two per cent of finely sifted dried blood was added to the 
soils in this series, or one gram per fifty grams of soil. In the 
case of the sandy soil three series in duplicate were tried, and the 
same relation under different conditions having been found to 
obtain between the ammonifying powers of the different organ- 
isms, we give only one duplicate set of the determinations. The 
incubation period was twelve days throughout at a temperature 
varying between 27° C and 30° C. The results of the ammoni- 
fication determinations with all the soils using dried blood 
throughout are given in Table III. The percentage of nitrogen 
of the total amount added which is made available is also given 
in every case. 

The data in Table III most strikingly indicate the .superiority 
of Ps. putida, B. vulgaris and Sarcina lutca to all other organisms 
in their efficiency at the production of ammonia from the nitrogen 
of dried blood. The next fact of singular interest is that B. 
mycoides, which has, in the hands of several investigators, shown 
such marked .superiority over other ammonifying organi.sms in 
the production of ammonia from organic nitrogen in solutions, 
manifests a relatively low power to transform the nitrogen of 
dried blood in soil cultures into ammonia. This, moreover, 
cannot be considered accidental, since different strains of B. 
mycoides, as above explained, exhibited that same lack of vigor in 
three duplicate sets of cultures run at different times and under 
slightly varying conditions of temiicratnro and period of incuba- 



1914] Lipmaii-Burgess : Ammoiiification in Soils by Pure Cultures 



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146 University of California FuWcations in Agricultural Sciences [Vol. 1 

tion. Of the three organisms showing the highest elifieiency, as 
above indicated, in transforming the nitrogen of dried blood to 
annnonia, B. vulgaris appears in its turn to be the most vigorous, 
though the other two approach it closely and are about equal 
among themselves. 



(latj-Loani Soil 

"When the clay-loam soil is used as the medium with dried 
blood, marked differences are apparent in the effieieucy of all 
the organisms. The poorer air supply of the clay-loam soil, due 
to its fineness and much greater tenacity, are evidently inimical 
to ammonia production, even though the same source of nitrogen 
— dried blood — is supplied for the ammonification process. The 
largest amount of ammonia produced in this series was that by 
B. proteus vulgaris, and even that was little more than one 
quarter of the amoiint produced by B. vulgaris as above noted 
in the sandy soil. 

Moreover, the most efficient transformers of the nitrogen in 
dried blood in the sandy soil medium are not necessarily the 
same as those in the clay-loam soil. For example, in the case of 
the clay-loam soil B. proteus vulgaris is the most efficient 
ammonia producer with dried blood as ammonifiable material, 
while in the sandy soil the same organism manifests less than half 
the ammonifying efficiency of B. vulgaris. This latter organism, 
however, stands second to B. proteus vulgaris in efficiency in 
the clay-loam soil and yields 7.49 mgs. of ammonia nitrogen as 
against 9.10 mgs. produced by the last-named organism. There 
appear to be four other organisms which approach the efficiency 
of the two just discussed in the clay-loam soil in the following 
order: Mic. tetragenus, B. ramosus, Streptothrir, sp., and Ps. 
putida. While the latter does not compare in efficiency in the 
clay-loam soil with that shown by it in the sandy .soil, it still 
manifests a notable efficiency. Sarcina lutea, however, appears 
to have lost in the clay-loam soil the marked ammonifying power 
possessed by it in the sandy soil. 

Of course it must be again emphasized that the amnimts of 
ammonia produced by all the organisms in the clay-loaiii soil 



1914] Lipman-Burgess : Ammonification in Soils by Pure Cultures 147 

series are relatively so small that the margin allowable for safe 
comparison must of necessity be much decreased and therefore 
comparisons are more difficult. 



Clay-Adobe Soil 

Passing on to a study of the data obtained with the clay-adobe 
soil as a mediiim. we find again that he physical condition of the 
soil is a powerful factor in determining the amount of ammonia 
produced in soils by pure cultures of organisms possessed of 
ammonifying powers, if the fifteen different organisms used are 
a suitable criterion. This confirms the findings of J. G. Lipman 
in his long series of ammonification experiments with mixed cul- 
tures. For practical purposes, we may add that most of the 
bacteria ammonify dried-blood nitrogen equally well in the clay- 
adobe soil and in clay-loam soil, though there does appear to be 
a slight though consistently greater amount of ammonia produced 
in the first-named soil. Again, we find in the clay-adobe soil 
an organism which stands out as far superior to all others in 
ammonifying efficiency and again also it is not the same organism 
as manifested that superiority in the preceding soil. Wliile the 
duplicate determinations here do not agree as well as might be 
desired, they indicate amounts so much greater than the quan- 
tities of ammonia produced by the other organisms of the series 
that there can be no doubt of the marked and superior efficiency 
of B. tumescens as an ammonia producer from the nitrogen of 
dried blood in the clay adobe soil as a medium. Two other 
organisms appear to be in the second class in this series and they 
are Mic. tetragenus and B. mycoides. The first, it can be seen 
from Table III, occupied third place in efficiency in the clay 
loam soil, but the second has thus far been relatively inefficient. 
All the other organisms of the series do not manifest differences 
in efficiency of sufficient magnitude to warrant further comment, 
except that it is curious and interesting to note that the most 
efficient organism in the sandy soil is the least efficient organism 
in the adobe soil. 

It should be remarked here that the generally excellent agree- 
ment between duplicate determinations, as shown in the table. 



148 Univer.titii of California Puilications in Agricultural Sciciwrs [Vol. 1 

eliminates the fear that physical conditions in the individual 
cultures might operate to produce the rather marked effects noted.' 

In a comparison of the three soils it appears that the follow- 
ing organisms are among tho.se which show the highest efficiency 
in transforming the nitrogen of dried blood into ammonia : Ps. 
putida, Sarcina lutea, B. vulgaris, B. proteiis vulgaris. Mic. 
tetragenus, B. tumescens. Of these organisms only Mic. trim- 
genus shows a high efficiency in all three soils. Of the others 
B. vulgaris shows a high efficiency in both the sandy and adobe 
soils and Ps. putida in the sandy and clay-loam soil. The rest 
namely Sarcina lutea, B. proteus vulgaris and B. tumescens. are 
markedly efficient only in one soil each, namely, the sandy soil for 
the first, the clay-loam soil for the second, and the clay-adobe 
.soil for the third. It appears to us particularly worthy of note, 
also, that of the last three organisms named B. proteus vulgaris 
and B. tumescens each holds a pre-eminent position of efficiency 
in its own soil which no other organism of the fifteen has 
approached ; and even in the case of Sarcina lutea we find that 
it occupies a position in efficiency in its favorite soil which is 
second only to that of B. vulgaris and not very far behind the 
latter. This rather remarkable condition would hardly seem to 
be accidental and appears to us to indicate for certain organi.sms 
marked preferences for certain physical characteristics in media 
in which they are grown. 

It must be added here, too, that only six of the fifteen 
organisms .show marked ammonifying efficiency as regards the 
nitrogen of dried blood even when tested in three widely different 
soil types. The others vary but little from one another in all 
soils. Oddly enough, the organism which has shown the highest 
ammonifying efficiency because it maintained it through all soils, 
namely, Mic. tetragenus, has never before been looked ujion, s(i 
far as we are aware, as an important ammonifier. That it should 
be the only one of fifteen organisms tested which should be about 
equally efficient in all soils used is not unworthy of note. 

It is further significant to note here that B. mijcoid(s. which 
lias always been regarded as one of the most efficient soil organ- 
isms at ammonification, does not in our experiments show any 
unusual activity in that direction, at anv rate when dried lilood 



n)14] Lipman-Burgess : Ammonification in Soils by Pure Cultures 149 

is used as the amnidnifiable material, no matter what the soil 
medium. A possible exception to this statement may be found 
in the case of the adobe soil, in which B. mycoidcs seems to be 
superior to all but three or four of the fifteen organisms tested. 
Such superiority, under the circumstances noted, is probably of 
little significance. 



Series II. Experiments with Tankage 
This series was arranged in a manner similar to the preced- 
ing, the sandy, clay loam, and clay-adobe soils again being u.sed 
as media, but the ammonifiable material in this case was a high- 
grade tankage, the nitrogen content of which was 9.62 per cent. 
The results of the ammonia determinations were as shown in 
Table IV on page 150. 

Discussion of Series II 
The Sandy Soil 
When we study in the foregoing table, the ammonification of 
tankage by pure cultures of bacteria, and compare the results 
with those of Table III, we see at once some very striking differ- 
ences between the ability of the same micro-organisms to produce 
ammonia from tankage and from dried blood respectively. Not 
only do more of the organisms show a high efficiency in trans- 
forming the tankage nitrogen to ammonia, but the point of 
highest efficiency is not reached by the same organisms as before, 
others having taken their places in this series with sandy soil as 
the culture medium. For example, we find that B. mesentericus, 
which in the preceding series showed throughout an extremely 
low ammonifying efficiency even in the sandy soil, now manifests 
in the same culture medium, which, however, has tankage in 
place of blood added to it, the highest efficiency of all of the 
organisms tested. Indeed, it occupies a position of its own in 
that direction, much as does B. vulgaris in the sandy soil of the 
last series. The organism which approaches it most closely in 
the same medium is B. proteus vidgaris which, however, falls 25 
per cent short of producing the amount of ammonia yielded by 



150 University of California Publications in Agricultural Sciences [Vol. 1 



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1914] Lipman-Burgess: Ammonification in Soils hy Pure Cultures 151 

B. mesentericus. Then follow, not far behind B. proteus vulgaris, 
B. tmnescens, B. ramosus, and B. vulgaris in the order named, 
the latter being more than 331/3 per cent short of the efficiency 
exhibited by B. mesentericus. Nearly all of the other organisms 
fall more than 50 per cent short of the efficiency of the last- 
named organism under this set of circumstances, namely, sandy 
soil with tankage. There are, therefore, but five organisms out of 
the fifteen tested which can be adjudged distinctly efficient 
ammonifying organisms under these conditions. B. mycoides 
again exhibits a low efficiency, and not far different from, though 
slightly below, that manifested by it in the same soil in the dried- 
blood series. 

It must be added here that, with the exception of B. mesen- 
tericus, the efficient organisms in this part of Series II have also 
shown more or less marked efficiency in the preceding series. 



The Clay-Loam Soil 

Most of the organisms tested in the clay-loam soil seem to 
have found the latter a more congenial medium for ammonifica- 
tion with tankage than they did when dried blood was present. 
In this part of Series II, three of the organisms, namely, B. 
mesentericus, B. vulgatus, and B. tumescens, were not only the 
most efficient ammonifiers but also about equal in their ammoni- 
fying power. It appears, therefore, that so far as tankage is 
concerned B. mesentericus is an equally efficient ammonifier in 
the clay-loam and sandy soils. B. tumescens has previously 
established its pre-eminent position among the fifteen organisms 
as an ammonifier of dried-blood nitrogen in the adobe soil, but 
B. vulcjatus enters here for the first time as a markedly efficient 
ammonifier. 

Only slightly behind the three organi.sms just discussed in 
their ammonifying efficiency as regards tankage nitrogen in the 
clay-loam soil are, in the order named, B. suhtilis, B. mycoides, 
and B. vulgaris. While the latter has manifested its high 
efficiency in other series above described, the first two organisnLs 
named for the first time in the work thus far described show 
marked ammonifying ability. Only three organisms in this part 



152 Uitiversity of California Publications in Agricultural Sciences [Vol. 1 

of Series II have shown themselves to he really weak ammonifiers, 
and they are B. proteus vulgaris, B. icteroides, and Mic. tctra- 
genus. The first and the third of these, it will be remembered, 
have given evidence of marked efficiency under other circum- 
stances, but the second has thus far been throughout an organism 
of low efficiency. It is striking to note the much greater uni- 
formity which exists in this portion of Series II in the ammoni- 
fying powers of four-fifths of the organism.s tested than that 
which obtains in other parts of this series and of other series. 



The Clay-Adobe Soil 

Here again we find the great uniformity in ammonifying 
power between the larger number of bacteria tested which i.s 
characteristic of the foregoing section of Series II. On the other 
hand, the clay-loam soil seems to have been a more congenial 
medium than the adobe soil for the ammonification of tankage 
nitrogen, for larger amounts of ammonia are produced in it by 
the same organisms in the same period of incubation. 

Sarcina lutea shows the highest efficiency as an ammonitter 
in this part of the series, but is only slightly more efficient than 
B. iumcscens. The organism taking third place is B. mijcoides 
which, indeed, is not far behind the other two. Next in order of 
importance and still very efficient ammonifiers are B. sitbtiliit and 
Streptofhri.r, sp. The otlier organism.s are considerably weaker 
ammonifiers than tliose just mentioned and B. icieroidis again 
proves to be distinctly the weakest. Both Sarcina lulfa and 
B. tumescens have, as mentioned above under other circum- 
stances, plainly evidenced their high efficiency as annuonifiers 
and B. mycoides and B. suhiilis have taken similar positions with 
respect to tankage in the clay-loam soil. Sfrppfothrir, sp.. it will 
be remembered, has also shown a high ammonifying power before 
in the case of the day-loam soil when dried blood was used. 

Comparing the three soils in this series with the same ones 
in the preceding series, it appears quite clear that taking them 
by and large, ammonifying bacteria manifest a iinuh liighcr 
efficiency with high-grade tankage than with dried lilood under 
similar conditions. Likewise also, in Series II a small number of 



1914] Lipmaii-Burgess : Ammoniftcatioii in Soils hy Pure Cultures 153 

organisms mostly identical with those in Series I seem to mani- 
fest a distinetl.y high efficiency which the much larger number 
of the balance do not in most cases even approach. It is worthy 
of remark, moreover, that B. mycoides attains or rather ap- 
proaches in portions of Series II its position of prime importance 
which has thus far so generally been accorded it among ammoni- 
fying organisms. 

Series III. Cottonseed IMeal 

The cottonseed meal used in this series showed on analysis a 
nitrogen content eqiial to 5.5 per cent. The experiment in this 
series was otherwise conducted like those of Series I and II, 
2 per cent of cottonseed meal being used, or 1 gram per 50 grams 
of soil. The results are shown in Table III. 

Again the efficiency of the organisms tested is much greater 
in the sandy soil than in either the clay-loam or adobe soils. 

Sandy Soil 
In this part of Series III the organism of greatest ammonify- 
ing efficiency is B. tumescens. Those approaching it closely in 
efficiency are B. vulgatus, Sarcina lutea, and B. mycoides in the 
order named. B. ramosus takes fifth place and the next three 
organisms fall more than 33V3 per cent short of producing the 
amount of ammonia yielded by B. tuiiuscciis. They are B. 
meseniericus, B. megatherium, and B. proteus vulgaris. The 
balance of the organisms show only about half the efficiency of 
B. Uimescens and in one ca.se, B. icteroides, which has in all pre- 
vious serie.s shown a very low efficiency, only about 25 per cent 
of the maximum efficiency is manifested. Taken as a whole, the 
data given in Table III for the Anaheim sandy soil reflects 
favorably on cottonseed meal as a source of available nitrogen 
under the conditions named. Again, the greatest efficiency is 
manifested in this section of Series III by organisms which in all 
cases have shown high efficiency in other series above reported. 
As in the case of the heavy soil in the preceding series with 
tankage, B. mycoides also shows marked efficiency in the light soil 
when cottonseed meal is used. 



154 University of Califoniia Publications in Agricultural Sciences [Vol. 1 



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1914] Lipman-Burgess : Ammonification in Smls by Pure Cultures 155 

The Clay-Loam Soil 
The most notable thing in this part of Series III is as above 
intimated, the very low efficiency of all of the organisms tested. 
Indeed only two organisms manifest any notable activity as 
ammonifiers of nitrogen in cottonseed meal in the clay-loam soil 
as a medium. These two in the order of their importance are 
B. ramosiis and Streptothrix, sp. To these, in view of the dis- 
agreement of the duplicates as above shown, B. vulgaris may 
probably be added, and perhaps also B. vulgatus. The other 
organisms are all distinctly below the first two mentioned, and 
the lowest efficiency thus far noted is that exhibited by Mic. 
tetragemis in this soil. Considering the high efficiency of the 
latter organism in Series I, the results just discussed are puzzling. 

The Clay-Adobe Soil 

Even a casual glance at the data obtained in the clay-adobe 
soil as a medium indicates the distinct superiority of that medium 
to the clay loam soil for ammonification of the nitrogen of cotton- 
seed meal. Not only relatively but absolutely the data obtained 
show the production of much larger amounts of ammonia in this 
portion of the work. 

As is the case in the sandy soil with cottonseed meal as the 
ammonifiable material, B. iumescens .shows its distinct superiority 
to the other organisms as an ammonifier in the adobe soil. The 
next most efficient organism is B. subtilis, which, however, is con- 
siderably less efficient ; and the next two organisms, about as far 
below B. subtilis in efficiency as the latter is below B. iumescens, 
are B. ramosus and B. mycoides. The other organisms are all 
low in efficiency, though in nearly all cases absolutely better than 
the same organisms in the clay-loam soil. Thus far we find that 
B. subtilis shows itself markedly efficient, for the first time, in 
the clay-adobe soil with cottonseed meal. All the other organisms 
above named have manifested marked efficiency in some parts 
of the foregoing series. 

Looking at Series III as a whole it is interesting to note that 
the nitrogen of cottonseed meal seems to be made available 
through the activity of pure cultures of ammonifying bacteria. 



156 University of Calif ornia Publications in Agricultural Sciences [Vol. 1 

with much greater rapidity than has heretofore been believed. 
The next striking fact brought out in these results is the clearly 
indicated superiority of the clay-adobe soil to the clay-loam soil 
as a medium for the amnionification of the nitrogen in cottonseed 
meal. The third point worthy of mention in Series III is the fact 
that we find again in it. as in the preceding series, only a few 
of the fifteen organisms tested which show marked ammonifying 
efficiency. 

The culture of I's. fluoresce iis died in the midst of these 
investigations and was not replaced. 



Series IV. Fish Guano 

Fish guano, in accordance with the teachings of Voorhees and 
other agricultural chemists, has always been esteemed a good 
source of available nitrogen ; in the words of Voorhees," ' ' rank- 
ing in availability well up to blood and tankage." It seemed to 
us therefore of importance to compare in these pure culture 
studies fish guano with the otlier organic materials discussed 
above. Accordingly a series was started similar to those above 
described, except that li/i grams of finely sifted fish guano was 
the ammonifiable material used per 50 grams of soil. The fish 
guano used contained 8.63 per cent nitrogen. The re-sults 
obtained are shown in Table VI. 

The data in Table VI not only seem to confirm the opinion of 
Voorhees as above stated, if ammonia production by pure cul- 
tures may be taken as a criterion for determining the availability 
of fish guano, but they indicate in most striking fashion what 
was not shown in any of the foregoing series for the fertilizers, 
and particularly in the sandy soil, namely, the obliteration of 
the marked physiological diflierences obtaining in other series 
between the differelit organisms. Of the four nitrogenous fer- 
tilizers thus far discussed, fish guano seems to contain the form 
of nitrogen most generally ammonified by a large group of 
bacteria. 



1914] Lipman-Burgess : Ammonification in Soils 61/ Pure Cultures 



g W 51 cc w td W 50 td W td td td 



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]58 University of California Publications in Agricultural Sciences [Vol. 1 

The Sandy Soil 

The good ammonifying power manifested by most of the 
organisms tested in this soil with fish guano finds its maximum 
in the ease of B. vulgaris, which, it will be remembered, has 
already given evidence of notable ammonifying efSciency with 
other nitrogenous fertilizers. B. tumescens, likewise an organism 
with a record as an ammonifier well established, is a very close 
second to B. vulgaris in its efSciency at the ammonification of the 
nitrogen in fish guano. Four other organisms distinctly in the 
second class, and about alike in ammonifying efficiency in this 
part of Series IV, are, in the order of their efficiency, Ps. putida, 
B. vulgatus, B. ramosus, and Sarcina lutea. Peculiarly enoiigh, 
B. icteroides shows an ammonifying efficiency here which places 
it in the second class, a position which it has thus far never even 
remotely approached in the series of experiments above discussed, 
nor, for that matter, in other series which are described below. 
All other organisms in this part of Series IV are distinctly- in 
the third class, but nevertheles manifest notable ammonifying 
efficiency. 

In no other series of results have we obtained such .sharp 
lines of demarcation between the classes of organisms here tested, 
and arbitrarily grouped, in accordance with their respective 
powers of transforming organic nitrogen into ammonia. 

The Clay-Loam Soil 
The change in the physical condition of the soil from the sand 
to the clay loam shows a marked effect on the ammonifying 
power of the same organisms. Nevertheless we find, on the whole, 
the best set of results thus far obtained with the clay loam when 
the series with fish guano is studied. In a class by itself under 
these conditions is B. megatherium, which is markedly superior 
to all other organisms in this part of Series IV, except B. mescn- 
tericus, which is a close second. The organism, however, which 
takes third place, B. vulgaris, falls about 20 per cent short of 
attaining the efficiency of B. megatherium under these conditions. 
Distinctly lower in efficiency in this part of Series IV are. in 
the order of tlieir impnrlaiiiT. but only sliiihtly different from 



191-1] Lipmaii-Burgess : Ammoiiificatioii in Soils by Pure Cultures 159 

one another, B. vulgatus, Ps. putida, B. ramosus, and B. tumes- 
cens. All of these, however, fall more than 30 per cent short of 
attaining the ammonifying efficiency of B. megatherium under 
the conditions here considered. Two other organisms, B. subtilis 
and Sarcina lutea, fall 40 per cent short of the eiificiency of B. 
megatherium, and the others all fall far below even that figure. 
It is singular, here again, that the physical nature of the soil 
medium employed should so strikingly and so variously influence 
the efficiency of the ammonifying bacteria. The two organisms 
which are distinctly superior in ammonifying ability as regards 
the nitrogen of fish guano in the clay loam soil were only of 
moderate efSciency with the same form of nitrogen in the sandy 
soil. And again, the organism which in the latter soil was 
paramount in its position retreats in the clay-loam soil to third 
place. 

The Clay-Adobe Soil 

This is the first series of those we have thus far considered, 
as the data in Table VI shows, in which the clay-adobe soil proves, 
on the whole, to be inferior to the clay-loam soil. Seven organ- 
isms out of fourteen here show an extremely low ammonifying 
efficiency, and two others are by no means efficient organisms. 
That leaves five organisms in this group which may be considered 
of importance. Of these, B. vidgaris is the mo.st efficient, but 
B. tumescens is not far behind it. B. suhtilis belongs to the 
second class in this group and B. protcus vulgaris and B. 
mycoides to the third class. It is rather unfortunate that it was 
not possible in this part of Series IV to obtain better agreement 
between duplicate determinations. It would not appear to us, 
however, that the discrepancies in question militate agaio.st the 
justice of the conclusions above drawn. 

B. megatherium holds a very good place in the sandy and 
clay loam soils of Series IV and a fair place in the adobe soil. 
The same is even more strikingly true of B. vulgaris, and in a 
minor degree this is also true of B. tumescens. The other organ- 
isms do not manifest such consistent efficiency under the three 
widely varying soil conditions. 



160 Vniversity of California Publications in Agricultural Sciences [Vol. 1 

^Miscellaneous Series 

For the purpose of comparing sheep and goat manure as well 
as phosphatie guano with the other organic materials above 
described and with peptone, it was deemed of interest to obtain 
data exemplifying the ammonification in the same soil of all the 
different materials above used with the three additional ones just 
mentioned. The sandy soil was chosen for this series and when 
peptone (Witte) was used, 0.5 gram of it was added to 50 grams 
of soil. The peptone contained 14.14 per cent N. The sheep 
and goat manure (2.13 per cent N), owing to its low nitrogen 
content, was added to the extent of 3 grams per 50 grams of soil, 
and the phosphatie guano (3.96 per cent N) was also added at 
the rate of 3 grams per 50 grams of soil. The results obtained 
are recorded in Table VII. 

As was to be expected, the very available form of nitrogen 
in the peptone allows of the production of much larger quantities 
of ammonia than do the l&ss available forms of the other materials. 
The fineness of division and easy solubility of the peptone, as well 
as the form of nitrogen which it contains, doubtless have con- 
tributed to the results. The more or less uniform decomposition 
of it, however, by most of the organisms tested shows peptone to be 
unsuited, as has been claimed by other investigators, for ammoni- 
fication studies with pure or mixed cultures when the application 
of the data obtained, to field conditions, is contemplated. How- 
ever that may be, Sarcina lutea shows the highest efficiency at 
ammonifying peptone nitrogen and at least six other organisms 
approach it rather closely. Kelatively speaking, all but two of 
the organisms tested are efficient ammonifiers of peptone nitrogen. 
But their position with respect to peptone, as can be seen from 
Table VII, is no criterion as to their efficiency with respect to 
the other materials. 

One of the interesting facts about the ammonification of the 
sheep and goat manure by pure cultures is that only one organism 
showed ammonifying efficiency worthy of the name, and that was 
B. megatherium. The other organisms showed a very slight 
power only of ammonifying the nitrogen in it. Just why this 
large discrepancy should exi.st with respect to this manure 
between B. )ii( (jiilln riiiiii and the otlier orgaiiisiiis still remains 



1914] Lipman-Burgess : Ammonification in Soils iy Pure Cultures 

g tfl ►d CO td M td fo ca _w w _w _W ^ W 

a tE- § S g S a I 3 g I eg ,| ^ S ^ « 





-5 
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162 University of California Publications in Agricultural Sciences [Vol. 1 

to be explained. If, therefore, the readine.ss of the transformation 
of its nitrogen into ammonia by pure cultures of ammonifiers is 
to be taken as a criterion, sheep and goat manure must be 
adjudged to contain a relatively unavailable form of nitrogen. 
The most amazing evidence portrayed in Table VII is the 
very high availability of the nitrogen of bat guano, which is a 
phosphatic guano. While without question the fact of the large 
amount of the guano used, as well as its low content of nitrogen, 
preclude an accurate and wholly justifiable comparison of it with 
the other nitrogenous materials, one cannot help being struck 
by the large transformation of its nitrogen into ammonia which 
nearly all of the organisms tested can accomplish. In many 
ways, the transformation of the nitrogen of bat guano into 
ammonia resembles that of the transformation of peptone 
nitrogen. The differences between the ammonifying powers of 
the different organisms are, however, unquestionably more 
marked in the case of the bat guano. In its efficiency as an 
ammonitier of bat guano nitrogen, B. mycoides appears for the 
first time in all the series studied to be distinctly superior to all 
the other organisms tested. B. vulgaris easily takes second place. 
Not far behind, however, and about equal in efficiency, are B. 
megatherium, B. vulgatus, B. tumescens, and Mic. tetragenus. 
In the third class are B. ramosus, B. suhtilis, and Ps. putida, B. 
proteus vulgaris, and B. mesentericus. In the fourth class are 
Streptothrix, sp., Sarciiia lutea, B. icteroides, and Ps. fluorescens. 



A Comparison op the Relative Availabilities of the Organic 
Materials above Employed based on the Percentage 
op Nitrogen Contained in Them that vv^as 
Transformed to Ammonia 
Thus far we have been considering only the relative degrees of 
efficiency as ammonifiers of the different organisms among them- 
selves as respecting a "given organic form of nitrogen in a given 
soil. There is possible, however, a further very interesting study 
of the data above given as a basis. We refer to the percentage 
of nitrogen which is transformed in the different materials into 
ammonia so as to give them a relative rating as to availability as 



1914] Lipmait-Burgess : Atninonification in Smls by Pure Cultures 



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164 University of California Publications in AqriruUural Sciences [Vol. 1 

regards the work of pure eulture.s. The cokimns on availability 
of the various tables show a comparison in different soils of all 
the materials as attacked by the same organisms, and give the 
pereentagas of nitrogen in those materials which were trans- 
formed to ammonia. In Table VIII all of the columns indicating 
percentages of nitrogen of different fertilizers made available 
are brought together from the other tables and one is enabled to 
compare with much greater ease the different materials on the 
basis of availability in the same soil and with the same organism 
and in different soils, with different organisms. 

TJic Sanrhj Soil 

So far as this soil is concerned, the data in Table VIII 
indicate clearly the superiorit.y, from the point of view of the 
availability of its nitrogen, of cottonseed meal to the other organic 
nitrogenous fertilizers with which it is compared. Not only are 
the absolute amounts of ammonia produced in most cases larger 
from cottonseed meal nitrogen than from other forms, but there 
are more organisms of the fifteen tested which can vigorously 
ammonify this form of nitrogen. So that to illustrate, there are 
but five organisms which have shown the power to transform 
15 per cent or more of the nitrogen of dried blood into ammonia 
in the sandy soil. Under similar conditions there are ten 
organisms which hold such a record for cottonseed meal. Tank- 
age .shows itself to be the next important nitrogenous fertilizer 
to cotton.seed meal from the point of view of the availability of 
its nitrogen. Thus, comparing it with dried blood as above, we 
find that there are nine organisms which transform 15 per cent 
or more of the nitrogen of tankage into ammonia. No such high 
availability is obtained at all in the case of the fish guano. 

When we consider these nitrogenous fertilizers from the point 
of view of the transformation of 10 per cent or more of their 
nitrogen into ammonia, we find that there are thirteen of the 
fifteen organisms tested which possess that power as regards 
cottonseed meal nitrogen and another comes very close to that 
point. In the ease of tankage nitrogen there are thirteen organ- 
isms with a similar power. In the case of dried-blood nitrogen 



1914] Lipmnn-Burgess : Ammonification in Soils by Piire Cnltures 165 

there are but ten organisms which can accomplish that task, and 
but seven such in the case of fish-guano nitrogen. 

From the point of view of availability by pure cultures 
therefore in the sandy soil the four nitrogenous fertilizers are 
to be rated as follows: cottonseed meal, tankage, dried blood, and 
fish guano. The first two are nearly alike and are far superior 
to the last two, which are nearly alike, but much more different 
from each other than the first two. 

The greatest efficiency at ammonification manifested In* any 
organi.sm in the sandy soil is that of B. mesentericus with tankage 
nitrogen, which transforms 32.52 per cent of the nitrogen present 
into ammonia in twelve days. It should be noted in this con- 
nection also that absolutely higher amounts of ammonia are 
produced from tankage nitrogen than from any other form in 
the sandy soil, even if there are fewer organisms which attack 
it readily than there are in the case of the cottonseed meal. 



Tlie Clay-Loam Soil 

Conditions are evidently entirely different for ammonification 
in this soil. Not only is the ammonia production very low so 
far as all the fertilizers are concerned, but they no longer bear 
to one another the relation which obtained between them in the 
sandy soil. There are thus but few organisms which possess the 
power of transforming 10 per cent or more of the total nitrogen 
in any of the four fertilizers into ammonia in twelve days. In 
fact, there are none such in the case of the dried blood, only two 
such each in the cases of cottonseed meal and fish guano, and 
seven such in the case of the tankage. No organism attains to 
the production of ammonia equivalent to 13 per cent of the total 
amount present in the clay-loam soil regardless of the kind of 
fertilizer at its disposal. 

The tankage, however, is superior to the cottonseed meal in 
the clay loam and distinctly so as above indicated. The cotton- 
seed meal takes second place, the fish guano third place, and the 
dried blood is by far the poorest. Indeed, no organism was 
capable of producing an amount of ammonia in excess of 6.91 



166 University of California Publications in Agricultural Sciences [Vol. 1 

per cent of the total amount of dried-blood nitrogen furnished, 
and that occurred in only one ease, all the other organisms pro- 
ducing much less. 

The Claij-Adobe Soil 

Some very striking facts become apparent when the avail- 
ability of the four fertilizers in clay-adobe soil are considered. 
AA-Tiile on the whole dried-blood nitrogen is only slightly more 
efficiently transformed into ammonia than in the clay-loam soil, 
cottonseed meal and tankage, particularly the former, are more 
vigorously acted on in the clay-adobe soil by most of the organ- 
isms. Fish guano, while not markedly so, is none the less superior 
here again to dried blood. 

Again comparing the different fertilizers on the ba.sis of the 
amounts of their nitrogen transformed into ammonia by the 
organisms tested, we find that four organisms transform 10 per 
cent or more of the nitrogen in cottonseed meal into ammonia, 
a like number accomplish similar results in the case of tankage, 
and none succeeds in that direction in either fish guano or dried 
blood. While thus cottonseed meal and tankage appear alike, 
a study of table VIII reveals the superiority of the former in the 
larger absolute amounts of nitrogen which are transformed there 
than in the case of the latter. 

The first striking fact shown in Table VIII is the marked 
superiority of the sandy soil as a medium for ammonification by 
pure cultures. Likewise the added fact of its superiority as a 
medium for most of the organisms tested must be noted in this 
connection. The second point of great intei-est is the .surprising 
fact of the superiority of the clay-adobe soil to the clay-loam 
soil as a medium for ammonification. From its tenacious nature 
one would suppose the former type to be a much poorer medium 
for ammonification than the clay loam and yet it is di.stinctly 
superior to the latter as regards tankage and cottonseed meal. 
As regards fish guano, it is slightly inferior to the clay loam and 
again as regards the dried blood about equal to the clay loam 
or possibly slightly superior. 

Considering all the data given in Table VIII from all points 
of view, tankage must be given first place as regards the avail- 



Ifll4] Lipmaii-Bur(irxs: Jmmonificdlioii in Soils by Pure Cultures 167 

ability of its nitrogen, cottonseed meal easily takes second place 
and, owing to its superiority in the sandy soil, dried blood takes 
third place. Considering the superiority of the fish guano to 
the dried blood in the other soils, however, it is probably fairer 
to adjudge dried blood and fish guano of equal availability from 
the point of view of the transformation of their nitrogen into 
ammonia by pure cultures of ammonifying bacteria. 



General Discussion 

Several of the facts which have come to light in the investiga- 
tions above described demand a word of comment with respect 
to their general significance. First as regards the relative 
efficiencies at ammonification of the different organisms tested, 
we find that there is marked variation. Indeed it is difficult to 
find an organism among the fifteen tested which consistently 
stands as the best ammonifier regardless of the soil and the 
ammonifiable material employed. There are, however, one or two 
organisms which nearly approach .such a description. In other 
words, it appears that, viewing ammonification of organic 
nitrogen from the standpoint of pure cultures, every organism 
will do best with a definite combination of soil and organic 
matter. To be sure there are some organisms of those tested, even 
though they be in the minority, which are consistently weak 
anmiouifiers. B. icteroides and Ps. fluorescens serve to exemplify 
such. 

That B. mtjcoides is by no means always the most efficient of 
ammonifying bacteria as has heretofore been believed is clearly 
indicated above. On the other hand, it does possess and manifest 
marked superiority in certain eases. Thus, for example, while 
showing poor or mediocre ammonifying power in different soils 
with dried blood, tankage and fish guano, it manifests great vigor 
in the case of cottonseed meal and succeeds in making the record 
for the percentage of nitrogen transformed in the case of bat 
guano, in which it transforms to ammonia 36.06 per cent of the 
nitrogen present. 



168 Vniversity of CaUfortiia Publications in Agricultural Sciences [Vol. 1 

Comparing the organisms in any one given soil as a medium 
we find some interesting facts. In the sandy soil, for exami)lt;, 
with dried blood B. vulgaris is the most efficient ammonifier. 
making available or transforming into ammonia 24.36 per cent 
of the nitrogen present. With tanl^age, B. mesentericus shows 
the highest efficiency, transforming 32.52 per cent of the nitrogen 
present into ammonia. "With cottonseed meal, B. tumescens is 
paramount, yielding an amount of ammonia equivalent to 24.30 
per cent of the amount of nitrogen present. With fi.sh guano, 
B. vulgaris again manifests its superiority over the other organ- 
isms by changing 13.09 per cent of the nitrogen present into 
ammonia. When bat guano is used, B. mycoides stands distinctly 
superior to all others, as above shown, by transforming 36.06 per 
cent of the nitrogen present into ammonia. In the case of sheep 
and goat manure there is but one efficient organism and that is 
B. megatherium, which transforms 24.97 per cent of the nitrogen 
present into ammonia. Lastly when peptone is used Sarcina 
lutea stands pre-eminent, and when all materials are compared, 
regardless of whether they were used in all soils or not, the last- 
named organism makes the record for availability by transform- 
ing 41.98 per cent of the nitrogen present into ammonia. There 
are thus six organisms out of the fifteen which make records in 
one and the same soil but with different forms of organic matter. 
One of the six stands superior in the cases of two nitrogenous 
materials, namely dried blood and fish guano, and that is 
B. vulgaris. 

Comparing the same organisms with the same nitrogenous 
materials, above iised, except the last three named, in the clay- 
loam soil, we find that with dried blood B. proteus vulgaris is 
most efficient, transforming 6.9 per cent of the nitrogen present 
into ammonia. With tankage, B. tum,escens is pre-eminent and 
transforms 12.74 per cent of the nitrogen present into ammonia. 
With cottonseed meal, Streptothrix, sp., is superior, transforming 
12.84 per cent of the nitrogen present into ammonia ; and lastly 
with fish guano, H. megatherium is again pre-eminent, trans- 
forming 11.66 per cent of the nitrogen present into ammonia. 
We see again, therefore, that in one and the same soil, with foiir 
different materials, four different organisms make records for 



1914] Lipman-Burgess: Am mollification in Soils by Pure Cultures 169 

availability. To be sure, two of these organisms have shown 
themselves superior to all others in the sandy soil but with 
different materials. 

In the case of the clay-adobe soil we find that with dried 
blood B. tumescens stands at the top of the list and transforms 
9.41 per cent of the nitrogen present into ammonia. With tank- 
age in the same soil, Sarcina lutea is the most efficient ammonifier 
and transforms 12.44 per cent of the nitrogen present into 
ammonia. With cottonseed meal, B. tumescens is again superior 
to all others and transforms 18.20 per cent of the nitrogen present 
into ammonia; and finally with fish guano B. vulgaris again 
assumes the ascendency and transforms 8.64 per cent of the 
nitrogen present into ammonia. B. tumescens is very nearly as 
efficient in this latter case as B. vulgaris. We have thus seen 
that organisms which in all cases have shown their superiority 
in other soils are also very efficient in the clay-adobe soil. 

It is clear, therefore, that only about half of the fifteen 
organisms tested show greatest efficiency in some soil or with 
some form of organic matter. Scrutinizing more closely the 
efficiencies of each of these, we must concede to B. tumescens the 
paramount place among them, for it has stood pre-eminent, in 
five combinations of soil and fertilizer, out of fifteen, and has in 
addition been close to first place in several other instances. 

Comparing our findings with those of Marchal, the following 
critical statements must be made. First, that results of solution 
cultures are no criterion as to results to be obtained in soils. 
Secondly, that no two forms of organic nitrogen are attacked and 
ammonified with the same vigor by any one organism. Thirdly, 
that different soils will modify an organism's power to ammonify 
any one given form of nitrogen very markedly, so that it may be 
efficient in one case and feeble in another. Fourthly, that the 
ammonifying efficiency of organisms is greater in sandy soil, and 
possibly in others, than in solutions, for we have obtained a 
transformation of 41.98 per cent of peptone nitrogen and 36.06 
per cent of bat guano nitrogen into ammonia by Sarcina lutea 
and B. mxjcoicles respectively in twelve days at temperatures 
between 27° C and 30° C, while Marchal only obtained similar 
transformations in thirty days at 30° C in albumin solutions. 



170 University of California Publications in Agricultural Sciences [Vol. 1 

Owing to the general distribution of the efficient ammonifying 
organisms above described in most soils, it is extremely improb- 
able, to judge from our results, that we may look forward 
to a profitable form of inoculation of soils with ammonifying 
bacteria. The choice, however, of the form of nitrogenous fer- 
tilizer for a .soil, which will be most readily made available, when 
available nitrogen is needed, may indeed be something of much 
greater practical significance, as our data would appear to 
indicate. Particularly emphatic are our results in illustrating 
that, at least so far as pure cultures are concerned, and as also 
partly shown by J. G. Lipman^ and others, with mixed cultures, 
prevalent ideas with respect to the relative availability of common 
nitrogenous fertilizers are incorrect. Both tankage and cotton- 
seed meal, and, in some cases, fish guauo, show higher avail- 
abilities than dried blood, which we have always been in the 
habit of regarding as the most available of organic nitrogenous 
fertilizers (based only on vegetation tests). 

Nor do we argue too far afield from our subject when we 
make the remarks just preceding. We recognize fully that 
availability as measured by ammonification does not necessarily 
denote availability as measured by nitrification nor by assimila- 
tion of nitrogen by plants. We cannot, however, help noting, 
also, that a persistent preference exists among practical orchard- 
ists in this state for tankage as against dried bood and we 
therefore feel that, judged by other critera, similar conclusions, 
must be drawn to those adduced from our experiments with pure 
cultures of ammonifying bacteria. 

We have decided, however, to go much further in these experi- 
ments and are now prosecuting more elaborate investigations on 
nitrogen transformation not only to ammonia but also to nitrates. 
In these studies we shall deal with the soil flora as existing in a 
large variety of soil types freshly collected from the field for our 
purposes. Moreover, we shall employ the raw, unsifted fertilizer 
material just as it is used by the farmer. From these investiga- 
tions we hope to glean much more evidence which will be of 
great practical significance, and, like the results above described, 
also of marked scientific interest. 



1914] Lipman-Btirgess : Ainmonifiration in Soils by Pure Cultures 171 

Summary 
Results ai'e above given which deal with : 

1. The marked differences in ammonifying efficiency of fifteen 
organisms in pure culture. 

2. The soil was used as a medium and three types employed, 
sandy, clay loam and clay adobe. 

3. Pour common fertilizers as sources of nitrogen were used, 
in all soils, and peptone, bat guano, and sheep and goat manure 
employed only in the sandy soil, besides. The four fertilizers 
were dried blood, tankage, cottonseed meal and fish guano. 

4. The nature of the soil, as well as the nature of the nitro- 
genous material, markedly modify an organism's ammonifying 
power. 

5. There is no regularity in these variations and they cannot 
be foretold. 

6. While it is difficult to make an exact decision, B. tnmescens 
appears, on the whole, to have been the most efficient organism 
above tested. 

7. The highest efficiency in a single culture with a fertilizer 
was shown by B. mrjcoides, which transformed in twelve days at 
27° to 30° C, 36.06 per cent of the nitrogen in bat guano into 
ammonia. 

8. The highest efficiency in a single culture with peptone was 
shown by Sarcina lutea, which transformed 41.98 per cent of the 
nitrogen present into ammonia under similar conditions. 

9. A comparison of availability of nitrogenous fertilizers with 
ammonifiability as a criterion, according to our experiments, 
markedly changes the positions of tankage, fish guano, and 
cottonseed meal with respect to dried blood, showing them in 
most or in many cases to be superior to the latter. 

10. One half of the number of organisms tested is far superior 
to the other half in ammonifying ability. 

11. Interesting comparisons with ilarchal's work are made. 

12. B. icteroides has shown itself throughout to possess but 
little ammonifying efficiency. 



172 Vnivcr.siti) of Californiii Publieationa iit Ayricnltural Sriences [Vol. 1 

13. Experiments are following those above described to make 
the latter more complete. Elaborate tests are being made witti 
many soil types using the original soil 's mixed flora from freshly 
collected samples in the field. Not only ammonifying powers of 
these mixed flora in the different soil types will be studied, but 
also the corresponding nitrifying powers. A large variety of 
nitrogenous fertilizers as employed in practice will be tested here. 



REFERENCES OP MORE OR LESS DIRECT PERTINENCE 

1 Bull. Acad. Roy. Soc. Belg., 3 ser., vol. 2.5, p. 727. 

2 Voorhees, Fertilizers, Maemillan Co., 1898. 

3 N. J. Sta. Bulletin, no. 246. 

4 Ann. Sci. Agron., vol. 19, p. 209. 

5 Rpt. Del. Exjx Sta., 1899, p. 76. 

6 Cent, f iir Bakt., 2" Abt., vol. 20, p. 322. 
^ Ibid., vol. 15, p. 433. 

s Ibid., vol. 29, p. 238. 
Ibid., vol. 31, p. 49. 



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li. Some Unreported Alaska Spbagna, together with a Summary of the 
Cryptogamic Work of the University of California Botanical Ex- 
pedition to Alaska in 1899, by William Albert Setchell. Pp. 309- 
315. September, 1907 05 

16. On Nutrient and Balanced Solutions, by W. J. V. Osterbout. Pp. 317- 

318. October, 1907 „ 05 

16. A Synopsis of the North American Godetias, by Willis Linn Jepson. 

Pp. 319-354; plate 29. December, 1907 40 

Indes, pp. 355-360. 
Vol. 3. 1. Compositae of Southern California, by Harvey Monroe Hall. Pp. 1- 

302; plates 1-3, with a map. December, 1907 3.00 

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amplexicaulis Nutt., by H. D. Deusmore. Pp. 303-330; plates 4-8. 
December, 1908 _ 35 

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ceous Corallines, I, by Maurice Barstow Nichols. Pp. 341-348; 
plate 9. December, 1908 _ _ 10 

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ceous Corallines. II, by Maurice Barstow Nichols. Pp. 349-370; 
plates 10-13. April, 1909 „ 15 

7. New Chlorophyceae from California, by Nathaniel Lyon Gardner. Pp. 

371-375; plate 14. April, 1909 _ 10 

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May, 1909 _ 16 

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1-74; plates 1-11; 15 text-figures. March, 1910 „ .76 

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Lyon Gardner. Pp. 121-136; plates 16-17. August, 1910 „ .16 

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159-176; plates 21-24. March, 1911 16 

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July, 1911 15 

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227. March, 1912 ^0 

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Pp. 269-281. June 26, 1912 ~ 15 

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Frionitis, by Lyman L. Daines. Pp. 283-302; plates 32-34. March, 

1912 20 

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plate 35. March, 1913 ~ 10 

18. New Fucaceae, by Nathaniel L. Gardner. Pp. 317-374; plates 36-53. 

April, 1913 V 75 

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Pp. 375-388. June, 1913 15 

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